7-alkylidene cephalosporanic acid derivatives and methods of using the same

ABSTRACT

Derivatives of 7-alkylidene cephalosporanic acid sulfone and the pharmaceutically active salts thereof are found to be potent inhibitors of β-lactamase enzymes.

This application is a divisional of U.S. patent application Ser. No.08/354,850, filed Dec. 9, 1994 now U.S. Pat. No. 5,629,306.

This invention was made with Government support under Contract No.5R01-GM-37774, awarded by the National Institutes of Health. TheGovernment has certain rights to the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel compounds which are β-lactamaseinhibitors, pharmaceutical compositions containing the same and methodsof inhibiting β-lactamases. More specifically this invention isconcerned with novel 7-alkylidene cephalosporins and pharmaceuticallyacceptable salts thereof.

2. Description of the Background

The most important mechanism of microbial resistance to β-lactamantibiotics is the bacterial production of β-lactamases, enzymes whichhydrolytically destroy β-lactam antibiotics, such as penicillins andcephalosporins. This type of resistance can be transferred horizontallyby plasmids that are capable of rapidly spreading the resistance, notonly to other members of the same strain, but even to other species. Dueto such rapid gene transfer, a patient can become infected withdifferent organisms, each possessing the same β-lactamase.

β-lactamase enzymes have been organized into four molecular classes: A,B, C, and D based on amino acid sequence. Class A, which includes RTEMand the β-lactamase of Staphylococcus aureus, class C, which includesthe lactamase derived from P99 Enterobacter cloacae, and class D areserine hydrolases. Class A enzymes have a molecular weight of about 29kDa and preferentially hydrolyze penicillins. The class B lactamases aremetalloenzymes and have a broader substrate profile than the proteins inthe other classes. Class C enzymes include the chromosomalcephalosporinases of gram-negative bacteria and have molecular weightsof approximately 39 kDa. The recently recognized class D enzymes exhibita unique substrate profile which differs significantly from both class Aand class C.

The class C cephalosporinases, in particular, are responsible for theresistance of gram-negative bacteria to a variety of both traditionaland newly designed antibiotics. The Enterobacter species, which possessa class C enzyme, are now the third greatest cause of hospital-acquiredinfections in the United States. This class of enzymes often has pooraffinities for inhibitors of the class A enzymes, such as clavulanicacid, a commonly prescribed inhibitor, and to common in vitroinactivators, such as 6-β-iodopenicillanate.

One strategy for overcoming rapidly evolving bacterial resistance is thesynthesis and administration of β-lactamase inhibitors. Frequently,β-lactamase inhibitors do not possess antibiotic activity themselves andare thus administered together with an antibiotic. One example of such asynergistic mixture is “augmentin which is a registered trademark of theSmithkline Beechom Corporation”, which contains the antibioticamoxicillin and the β-lactamase inhibitor, clavulanic acid.

It is thus desirable to find novel β-lactamase inhibitors which can becoadministered with a β-lactam antibiotic.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide novelβ-lactamase inhibitors.

It is another object of the present invention to provide pharmaceuticalcompositions useful for inhibiting a β-lactamase.

It is another object of the present invention to provide pharmaceuticalcompositions with increased β-lactam antibiotic activity.

It is another object of the present invention to provide methods ofinhibiting a β-lactamase.

It is another object of the present invention to provide methods ofenhancing the biological activity of a β-lactam antibiotic.

These and other objects, which will become apparent during the followingdetailed description, have been achieved by the inventors' discoverythat compounds of the formula (1)

wherein n is 0 or 1 (the sulfide or the sulfone, respectively);

R₁ and R₂ are the same or different and are selected from the groupconsisting of

a) hydrogen;

b) linear or branched C₁₋₁₀-alkyl;

c) halogen;

d) hydroxy-C₁₋₁₀-alkyl;

e) C₁₋₁₀-alkoxy;

f) C₂₋₁₀-alkanoyloxy;

g) C₂₋₁₀-alkene;

h) C₂₋₁₀-alkene substituted with one or more groups selected from thegroup consisting of chlorine, fluorine, bromine or phenyl;

i) C₁₋₁₀-alkoxycarbonyl;

j) C₁₋₁₀-alkoxycarbamido;

k) N-C₁₋₁₀-alkoxy-N-C₁₋₁₀-alkylaminocarbonyl;

l) halo-C₁₋₁₀₋-alkyl;

m) C₆₋₁₀-aryl;

n) C₆₋₁₀-aryl substituted with one or more groups selected from thegroup consisting of ethyl, n-propyl, isopropyl, amino, methylamino anddimethylamino;

o) a C₂₋₁₀-heterocycle having from 1-3 heteroatoms selected from thegroup consisting of O, N and S; and,

p) —COOH or —COOY, wherein Y is pharmaceutically acceptable cation;

 R₃ is selected from the group consisting of

1) —COOH;

2) chlorine or fluorine;

3) trifluoromethyl;

4) —CHO; and,

5) —CH₂M where M is selected from the group consisting of

a) hydrogen;

b) halogen;

c) hydroxy;

d) C₁₋₁₀-alkoxy;

e) C₆₋₁₀-aryloxy;

f) C₆₋₁₀-aryl-C₁₋₁₀-alkoxy;

g) mercapto;

h) mercapto substituted with one or more groups selected from the groupconsisting of methyl, ethyl or phenyl;

i) C₂₋₁₀-acylthio;

j) C₂₋₁₀-acyloxy or carbamoyloxy;

k) C₂₋₁₀-acyloxy or carbamoyloxy substituted with one or more groupsselected from the group consisting of —COOH, aminophenyl, phenyl,C₁₋₆-alkyl, chlorine, bromine or fluorine;

1) a quaternary ammonium salt;

m) amino or amido; and,

n) amino or amido substituted with one or more groups selected from thegroup consisting of C₁₋₁₀-alkyl groups;

R₄ is selected from the group consisting of

a) hydrogen; and,

b) pharmaceutically acceptable cations; are effective β-lactamaseinhibitors.

DETAILED DESCRIPTION OF THE INVENTION

Thus, in a first embodiment, the present invention provides novelcompounds of the formula (1)

wherein n is 0 or 1;

R₁ and R₂ are the same or different and are selected from the groupconsisting of

a) hydrogen;

b) linear or branched C₁₋₁₀-alkyl, preferably, C₁₋₆-alkyl, morepreferably, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl,n-pentyl, cyclopropyl, cyclopentyl, or cyclohexyl, most preferablyt-butyl;

c) halogen, preferably Br or Cl;

d) hydroxy-C₁₋₁₀-alkyl, preferably, hydroxy-C₁₋₆-alkyl, more preferably,hydroxymethyl, 1-hydroxyethyl or 2-hydroxyethyl;

e) C₁₋₁₀-alkoxy, preferably, C₁₋₆-alkoxy, more preferably, t-butoxy ormethoxy;

f) C₂₋₁₀-alkanoyloxy, preferably, C₂₋₆-alkanoyloxy, more preferably,acetoxy or propanoyloxy;

g) C₂₋₁₀-alkene, preferably, C₂₋₆-alkene, more preferably, ethylene,1-propylene or 2-propylene;

h) substituted C₂₋₁₀-alkene, preferably, C₂₋₆-alkene, more preferablyethylene, 1-propylene or 2-propylene, wherein said substituents are onemore groups selected from the group consisting of chlorine, fluorine,bromine or phenyl;

i) C₁₋₁₀-alkoxycarbonyl, preferably, C₁₋₆-alkoxycarbonyl, morepreferably, methoxycarbonyl or t-butoxycarbonyl;

j) C₁₋₁₀-alkoxycacbamido, preferably, C₁₋₆-alkoxycarbamido, morepreferably, methoxycarbamido, ethoxycarbamido or n-propoxycarbamido;

k) N-C₁₋₁₀-alkoxy-N-C₁₋₁₀-alkylaminocarbonyl, preferably,N-C₁₋₆-alkoxy-N-C₁₋₆-alkylaminocarbonyl, more preferably,N-methoxy-N-methylaminocarbonyl, N-ethoxy-N-methylaminocarbonyl,N-methoxy-N-ethylaminocarbonyl or N-ethoxy-N-ethylaminocarbonyl;

1) halo-C₁₋₁₀-alkyl, preferably, halo-C₁₋₆-alkyl, more preferably,chloromethyl, 1-chloroethyl or 2-chloroethyl;

m) C₆₋₁₀-aryl group, preferably, phenyl, tolyl, anisoyl, mesityl, andxylyl;

n) substituted C₁₋₁₀-alkyl, preferably, phenyl, tolyl, anisoyl, mesityl,and xylyl, wherein said substituents are one or more groups selectedfrom the group consisting of ethyl, n-propyl, isopropyl, amino,methylamino and dimethylamino;

o) a C₂₋₁₀-heterocycle having from 1-3 heteratoms selected from thegroup consisting of O, N and S, preferably, triazolyl, triazinyl,oxazoyl, isoxazolyl, oxazolidinoyl, isoxazolidinoyl, thiazolyl,isothiazoyl, pyrazolyl, imidazolyl, pyrrolyl, pyrazinyl, pyridinyl,morpholinyl, quinolinyl, isoquinolinyl, indolyl, and pyrimidinyl, morepreferably, pyridinyl; and,

p) —COOH or —COOY, wherein Y is a pharmaceutically acceptable cation,preferably, sodium, potassium, calcium, or any other pharmaceuticallyacceptable cation known in the art;

 R₃ is selected from the group consisting of

1) —COOH;

2) Cl or F;

3) trifluoromethyl;

4) —CHO; and,

5) —CH₂M, wherein M is selected from the group consisting of

a) hydrogen;

b) halo, preferably F, Cl, Br, or I;

c) hydroxy;

d) C₁₋₁₀-alkoxy, preferably, C₁₋₆-alkoxy, more preferably, methoxy,ethoxy, n-propoxy or isopropoxy;

e) C₆₋₁₀-aryloxy, preferably, C₆₋₁₀-aryloxy, more preferably, phenoxy ornaphthoxy;

f) C₆₋₁₀-aryl-C₁₋₁₀-alkoxy, preferably, C₆₋₁₀-aryl-C₁₋₆-alkoxy, morepreferably, phenylmethoxy, 1-phenylethoxy or 2-phenylethoxy;

g) mercapto, preferably, thiol;

h) substituted mercapto, preferably, thiol, wherein said substituentsare selected from the group consisting of methyl, ethyl or phenyl;

i) C₂₋₁₀-acylthio, preferably C₂₋₆-acylthio, more preferably, acetylthioor propanoylthio;

j) C₂₋₁₀-acyloxy or carbamoyloxy, preferably, C₂₋₆-alkanoyloxy,C₆₋₁₀-aryl-carbonyloxy, carbamoyloxy or thiocarbamoyloxy, morepreferably, acetoxy or benzoyloxy;

k) substituted C₂₋₁₀-acyloxy or carbamoyloxy, preferably,C₂₋₆-alkanoyloxy, C₆₋₁₀-aryl-carbonyloxy, N-C₁₋₆-alkylcarbamoyloxy,N,N-di-C₁₋₆-alkylcarbamoyloxy, thiocarbamoyloxy,N-C₁₋₆-alkylthiocarbamoyloxy or N,N-di-C₁₋₆-alkylthiocarbamoyloxy, morepreferably, acetoxy, α-aminophenylacetoxy, benzoyloxy,benzyloxycarbonyloxy, succinoyloxy, N-methylcarbamoyloxy,N-ethylcarbamoyloxy, N,N-dimethylcarbamoyioxy, N,N-diethylcarbamoyloxy,N-methylthiocarbamoyloxy, N-ethylthiocarbamoyloxy,N,N-dimethylthiocarbamoyloxy, N,N-diethylthiocarbamoyloxy, wherein saidsubstituents are one or more groups selected from the group consistingof —COOH, aminophenyl, phenyl, methyl, ethyl, chlorine, bromine orfluorine;

1) a quaternary ammonium salt, preferably trimethyl ammonium chloride ortriethyl ammonium chloride;

m) amino or amido group, preferably —NH₂ or —CONH₂; and,

n) substituted amino or amido group, preferably —NH₂ or —CONH₂, whereinsaid substituents are one or two C₁₋₁₀-alkyl groups, preferablyC₁₋₆-alkyl groups, more preferably, methyl, ethyl, n-propyl, isopropylor n-butyl;

 R₄ is selected from the group consisting of

a) hydrogen; and,

b) pharmaceutically acceptable cations, preferably, sodium, potassium orcalcium.

In a preferred embodiment, n is 0, R₂ is hydrogen and R₁ is selectedfrom the group consisting of t-butyl, phenyl, pyridyl, COMe andN-methyl-N-methoxy-aminocarbonyl.

In a more preferred embodiment, n is 0, R₂ is hydrogen, and R₁ isselected from the group consisting of CO₂-t-Bu and CHO.

In another preferred embodiment, n is 1, R₂ is hydrogen, and R₁ isselected from the group consisting of CO₂Me, CH₂OH and t-butyl.

In another more preferred embodiment, n is 1, R₂ is hydrogen and R₁ isselected from the group consisting of phenyl, pyridyl and CO₂-t-butyl.

In another preferred embodiment, n is 0 and R₁ and R₂ are the same andare selected from the group consisting of bromine and chlorine.

In another preferred embodiment, n is 1 and R₁ and R₂ are the same andare selected from the group consisting of bromine and chlorine.

In another preferred embodiment, n is 0, R₁ is hydrogen, and R₂ isbromine.

In another preferred embodiment, n is 1, R₁ is hydrogen, and R₂ isselected from the group consisting of phenyl and bromine.

In a most preferred embodiment, n is 1, R₁ is pyridyl, R₂ is hydrogen,R₃ is —CH₂OAc and R₄ is sodium.

Compounds according to formula (1) were obtained as follows.7-Aminocephalosporanic acid (commercially available from Aldrich),esterified with diphenyl diazomethane (2), was treated with excesstriethylamine and trifluoromethanesulfonic anhydride and the resultanttrifluorosulfonyl imine was hydrolyzed to produce benzhydryl7-oxocephalosporanate 3. (See Hagiwara, D. F.; Sawada, K.; Ohnami, T.;Aratani, M.; Hashimoto, M. J. Chem Soc. Chem. Commun. 1982, 578.) Due toits instability, 3 was used directly in the next step withoutpurification.

The 7-alkylidenecephalosporanates 4 were prepared by treating7-oxocephalosporanate 3 with the corresponding Wittig reagent at −78° C.Compounds 4 a-k were prepared in the standard manner with the exceptionsof 4b, 4j, 4l, and 4m. Compound 4b required the addition of the Zn/Cucouple to 3 in the presence of CCl₄ and PPh₃ to lead to its formation.Compound 4j was prepared by the reduction of 4i with NaCNBH₃. Compound4a was reduced by the Zn/Cu couple to produce monobromomethylenecephem4l, which was further treated with t-BuLi and CuCN to give compound 4mas shown below.

no. R₁ R₂ 4a Br Br 4b Cl C1 4c H Ph 4d Ph H 4e Py H 4f CO₂CH₃ H 4gCO₂C(CH₃)₃ H 4h COCH₃ H 4i CHO H 4j CH₂OH H 4k CON(CH₃)(OCH₃) H 4l H Br4m t-butyl H

Many of the compounds in the series 4 were oxidized with excess m-CPBAyielding the corresponding sulfones 5.

Deprotection of compounds 4 and 5 gave the corresponding sodium salts 6and 7 as shown below.

no. R₁ R₂ 6a Br Br 6b Cl Cl 6e Py H 6f CO₂CH₃ H 6g CO₂C(CH₃)₃ H 6h COCH₃H 6i CHO H 6k CON(Me)(OMe) H 6l H Br 6m t-Bu H 7a Br Br 7b Cl Cl 7a H Ph74 Ph H 7e Py H 7g CO₂C(CH₃)₃ H 7j CH₂OH H 7l H Br 7m t-Bu H

Compounds containing R₁ and R₂ groups (i.e., alkoxy or alkene) not shownabove may be synthesized by using an appropriate Wittig reagentR₁R₂C═PP₃. The Wittig reagents ROCH═PPh₃ and H₂C═CH—CH═PPh₃ may be usedto make the 7-alkyoxymethylene and 7-alkenylmethylene compounds,respectively.

In addition to the Wittig reaction, the Peterson olefination proceduremay be used to form 7-substituted methylene compounds from theoxocephalosporanate 3. For example, (RO) (SiMe₃)CHLi or(haloalkyl)(SiMe₃)CHLi may add to 3 to form the 7-alkoxymethylene or7-halomethylmethylene compounds, respectively.

The 7-alkanoylmethylene species may be made by forming the vinyl anionand reacting it with a desirable alkanoyl halide. The vinyl anion may bemade by a standard lithium-halogen (or magnesium-halogen) exchangereaction, for example, reaction of 4a with methyl lithium. The lithiumvinyl group may then be functionalized by reaction with analkoxycarbonyl chloride.

The 7-carboxylmethylene compounds (R₁ or R₂=COOH or COOY) may be formedby hydrolysis of the corresponding ester, preferably, the correspondingt-butyl ester.

The compounds wherein R₃ is a halogen may be formed by displacement ofthe —OAc group with ethylxanthate (EtOCS₂K). Raney-Nickeldesulfurization (H₂/Ra-Ni) would yield the exocyclic alkene which maythen be ozonized to the 3-hydroxy cephem. Reaction with a halogenatingreagent would provide the 3-halo species. For example, PCl₅ may be usedto convert the 3-OH group into a 3-Cl group. The 3-methyl species may beobtained by the rearrangement of the exocyclic alkene, formed byRaney-Nickel desulfurization, by reaction with Et₃N. The 3-hydroxymethylspecies may be obtained by hydrolysis of the —OAc group with NaOH or anappropriate enzyme. The 3-halomethyl species may be formed by reactionof the 3-hydroxymethyl species with a halogenating reagent. For example,PCl₅ may be used to form the 3-chloromethyl species.

The compounds wherein M is alkoxy, aryloxy, or arylalkoxy may beobtained by reaction of the 3-hydroxymethyl species with tosyl chlorideand displacement of the resultant tosylate with an oxide. For example,sodium methoxide may be used to obtain the 3-methoxymethyl species. Thecompounds wherein M is mercapto may be formed by reaction of the3-chloromethyl compound with sodium sulfhydride (NaSH). This compoundmay further be derivatized with an alkylhalide to form a substitutedmercapto or an acylchloride to form an acylthio group.

The species wherein M is an amino group may be formed by the GabrielSynthesis, i.e., reaction of the 3-chloromethyl compound with potassiumphthalimide and hydolysis of the product with acid to yield the3-aminomethyl compound. The 3-ammoniomethyl compound may be formed byreaction of the 3-aminomethyl compound with methyl chloride to form the3-trimethylammoniomethyl chloride.

The compound wherein M is an amido group (CONH₂) may be formed bydisplacement of the tosylate described above with cyanide, e.g. KCN,followed by hydrolysis of the resulting nitrile to the amide.

The aforementioned salts of 7-alkylidene cephems were evaluated asinhibitors of the Class C β-lactamase of Enterobactor cloacae P99 andTEM2 by relative IC₅₀ analysis. The IC₅₀ value represents theconcentration of inhibitor required to effect a 50% loss of activity offree enzyme. The IC₅₀ value of each compound was determined as follows.Following a 10 minute incubation of a dilute solution of enzyme (2.56nM) and inhibitor (<0.64 mM), a 50 mL aliquot of this incubation mixturewas then further diluted into 1 mL nitrocefin solution, and the rate ofhydrolysis was measured during a 1 minute period by monitoring theabsorbance of nitrocefin as a function of time. In addition, the IC₅₀values of tazobactam and clavulanic acid were determed as relativecontrols. The data is presented in Table 1 below.

TABLE 1 E. cloacae P99 and SC 12368, and E. coli. W3310 β- lactamaseinhibitory activity IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) E. cloacea E. coli E.cloacea Compound n R₁ R₂ P99 WC3310 SC12368 tazobactam 943 25 4000Clavulanic >20000 60 >20000 acid 6a 0 Br Br >20000 >20000 >20000 6b 0 ClCl >20000 >20000 >20000 6d 0 Ph H >20000 >20000 >20000 6e 0 2′-PyH >20000 >20000 >20000 6g 0 CO₂C(CH₃)₃ H 2500 >20000 >20000 6h 0 COCH₃H >20000 >20000 >20000 6i 0 CHO H 8200 >20000 16500 6k 0 CON(CH₃)-H >20000 >20000 >20000 (OCH₃) 6l 0 H Br >20000 >20000 >20000 6m 0 t-BuH >20000 >20000 >20000 7a 1 Br Br >20000 >20000 >20000 7b 1 Cl Cl >200008300 >20000 7c 1 H Ph >20000 >20000 >20000 7d 1 Ph H 6250 >20000 6800 7ei 2′-Py H 25 800 25 7f 1 CO₂CH₃ H >20000 8 >20000 7g 1 CO₂C(CH₃)₃ H 78005 5900 7j 1 CH₂OH H >20000 >20000 >20000 7l 1 H Br >20000 >20000 >200007m 1 t-Bu H >20000 >20000 >20000

Compound 7e, 7-(Z)-[(2-pyridyl)methylene] cephalosporanic acid sulfone,was determined to be more potent than tazobactam, showing a 20 foldincrease in activity. In general, it was found that the sulfones weremore potent than their corrosponing sulfide analogs. One strikingexample of this is the 1300 fold increase in activity of 7e, the pyridylsulfone, over it's sulfide 6e.

In a second embodiment, the present invention provides pharmaceuticalcompositions useful for inhibiting a β-lactamase. The presentpharmaceutical compositions comprise at least one of the present7-alkylidene cephalosporins and at least one pharmaceutically acceptablecarrier.

The present compositions may be employed in capsule form or as tablets,powders or liquid solutions or as suspensions or elixirs. They may beadministered orally, intravenously or intramuscularly. The presentcompositions are preferably presented in a form suitable for absorptionby the gastro-intestinal tract.

Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional pharmaceutical carrierssuch as binding agents, for example, syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrrolidone; fillers, for example, lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine, lubricants,for example, magnesium stearate, talc, polyethylene glycol, silica;disintegrants, for example, potato starch; or acceptable wetting agentssuch as sodium lauryl sulphate. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may be in theform of aqueous or oily suspensions, solutions, emulsions, syrups,elixirs, etc. or may be presented as a dry product, for reconstructionwith water or other suitable vehicle before use. Such liquidpreparations may contain conventional additives such as suspendingagents, for example, sorbitol syrup, methyl cellulose, glucose/sugarsyrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminumstearate gel or hydrogenated edible fats; emulsifying agents, forexample lecithin, sorbitan monooleate or acacia; non-aqueous vehicles,which may include edible oils, for example, almond oil, fractionatedcoconut oil, oily esters, propylene glycol, or ethyl alcohol;preservatives, for example, methyl or propyl p-hydroxybenzoates orsorbic acid. Suppositories will contain conventional suppository bases,e.g., cocoa butter or other glyceride.

Compositions for injection may be presented in unit does form inampules, or in multidose containers with an added preservative. Thecompositions may take such forms as suspensions, solutions, emulsions inoily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for reconstitution with asuitable vehicle, e.g., sterile, pyrogen-free water, before use.

The present compositions may also be prepared in suitable forms forabsorption through the mucous membranes of the nose and throat orbronchial tissues and may conveniently take the form of powder or liquidsprays or inhalants, lozenges, throat paints, etc. For medication of theeyes or ears, the preparations may be presented as individual capsules,in liquid or semi-solid form, or may be used as drops, etc. Topicalapplications may be formulated in hydrophobic or hydrophilic bases asointments, creams, lotions, paints, powders, etc.

Also, in addition to a carrier, the present compositions may includeother ingredients such as stabilizers, binders, antioxidants,preservatives, lubricators, suspending agents, viscosity agents orflavoring agents and the like.

For veterinary medicine, the composition may, for example, be formulatedas an intramammary preparation in either long acting or quick-releasebases.

The dosage to be administered depends to a large extent upon thecondition of the subject being treated and the weight of the subject,the route and frequency of administration, the parenteral route beingpreferred for generalized infections and the oral route for intestinalinfections.

The instant compositions may be administered in several unit dosageforms as, for example, in solid or liquid orally ingestible dosage form.The compositions per unit dosage, whether liquid or solid may containfrom 0.1% to 99% of active material (the present 7-alkylidenecephalosporins and optional antibiotic), the preferred range being fromabout 10-60%. The composition will generally contain from about 15 mg toabout 1500 mg by weight of active ingredient based upon the total weightof the composition; however, in general, it is preferable to employ adosage amount in the range of from about 250 mg to 1000 mg. Inparenteral administration the unit dosage is usually the pure compoundin a slightly acidified sterile water solution or in the form of asoluble powder intended for solution.

The present β-lactamase inhibitors will be particularly useful in thetreatment of infections caused by Enterobacter, Citrobacter, andSerratia. These bacteria have the ability to attach to the epithelialcells of the bladder or kidney (causing urinary tract infections) andare resistant to multiple antibiotics including amoxicillin andampicillin. The present β-lactamase inhibitors would also be useful inthe treatment of infections caused by highly resistant Pneumococci. Suchdiseases include otitis media, sinusitis, meningitis (both in childrenand adults), bacteremia, and septic arthritis. Resistant pneumococcalstrains have surfaced in many parts of the world. For example, inHungary, 58% of S. pneumoniae are resistant to penicillin, and 70% ofchildren who are colonized with S. pneumoniae carry resistant strainsthat are also resistant to tetracycline, erythromycin,trimethoprin/sulfamethoxazole (TMP/SMX), and 30% resistant tochloroanphenicol. Klebsiella pneumoniae (resistant via the production ofβ-lactamase) have caused hospital outbreaks of wound infection andsepticemia.

Thus, in a third embodiment, the present invention providespharmaceutical compositions with increased β-lactam antibiotic activity.This pharmaceutical composition is as defined above, but in addition toat least one of the present 7-alkylidene cephalosporins and at least onea pharmaceutically acceptable carrier, the compositions also contains atleast one β-lactam antibiotic. The β-lactam antibiotic may be any of theabove-noted antibiotics or any other known in the art, preferablyamoxicillin or piperacillin, and its selection will depend upon whatindication is necessary.

In a fourth embodiment, the present invention provides a method ofinhibiting a β-lactamase, comprising administering to a patient in needthereof an effective amount of at least one of the present 7-alkylidenecephalosporins. The method of administration may be any of theabove-noted methods or any other known to one of skill in the art.

In a fifth embodiment, the present invention provides a method ofenhancing the biological activity of a β-lactam antibiotic bycoadministering to a patient in need thereof, an effective amount of oneof the present 7-vinylidene cephalosporins and an effective amount of atleast one β-lactam antibiotic. The method of administration may be anyof the above-noted methods or any other known to one of skill in theart. The β-lactam antibiotic may be any of the above-noted β-lactamantibiotics or any other known in the art.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLES Example 1 Benzhydryl 7β-aminocephalosporanate (2)

To a suspension of 7-aminocephalosporanic acid (130.4 g, 0.48 mol) inmethanol (480 mL) was added a solution of diphenyldiazomethane (93.0 g,0.48 mol) in CH₂Cl₂, prepared by the oxidation of benzophenone hydrazoneby mercury oxide at room temperature. The reaction was then mechanicallystirred at room temperature for 44 hours. The remaining solid wasremoved by filtration. The resultant filtrate was concentrated in vacuoand purified by column chromatography (10% CH₃OH in CH₂Cl₂) to affordthe desired ester as pale yellow solid (86.1 g, 41% yield). R_(f)=0.44in 1:9 CH₃OH:CH₂Cl₂, mp. 45-46° C.; IR (CHCl₃) 2980, 1780, 1730 cm⁻¹; ¹HNMR (CDCl₃) δ 8.41 (2H, bs), 7.22 (10H, m), 6.91 (1H, s), 5.27 (1H, d,J=2.8 Hz), 5.15 (1H, d, A of ABq, J=14 Hz), 4.94 (1H, s), 4.84 (1H, d, Bof ABq, J=14 Hz), 3.73 (1H, d, A of ABq, J=17 Hz), 3.33 (1H, d, B ofABq, J=17 Hz), 1.92 (3H, s); ¹³C NMR (CDCl₃) δ 169.8, 168.8, 160.6,138.9, 138.7, 129.5, 129.3, 129.1, 128.7, 128.5, 127.97, 127.61, 127.52,127.18, 126.52, 126.06, 125.4, 79.0, 63.3, 62.6, 58.5, 25.7, 20.1.

Example 2 Benzhydryl 7-oxocephalosporanate (3)

The title compound was prepared by modifying the procedure of Haqiwaraet al. To a solution of benzhydryl 7β-aminocephalosporanate, (5.9 g,13.5 mmol) in anhydrous CH₂Cl₂ (70 mL) at −78° C., triethylamine (5.6mL, 40.4 mmol) was added dropwise with stirring. After 5 minutes,trifluoromethanesulfonic anhydride (6.8 mL, 40.4 mmol) was addeddropwise to this solution over a 5 minute period. The reaction mixturewas allowed to warm slowly to 0° C. over a 1 hour period. It was thenrecooled to −78° C. and triethylamine (5.6 mL, 40.4 mmol) was added overapproximately 10 minutes. The reaction mixture was stirred at −78° C.for an additional 30 minutes and poured into 200 mL cold 0.5 N HCl. Theresultant mixture was further stirred until the ice melted. The layerswere separated and the aqueous layer was extracted with CH₂Cl₂ (150 mL).The combined organic layers were washed with cold 0.5 N HCl (3×100 mL),dried (Na₂SO₄), and concentrated (at room temperature or below) toproduce the title compound (5.8 g, 98% yield) as a brown solid which wasused without further purification. IR (CHCl₃) 3005, 1830, 1790, 1740cm⁻¹; ¹H NMR (CDCl₃) δ 7.39 (10H, m), 7.05 (1H, s), 5.32 (1H, s), 5.07(1H, d, A of ABq, J=14 Hz), 4.85 (1H, d, B of ABq, J=14 Hz), 3.64 (1H,d, A of ABq, J=18 Hz), 3.44 (1H, d, B of ABq, J=18 Hz), 2.05 (3H, s);¹³C NMR (CDCl₃) δ 188.4 (s), 170.3 (s), 160.1 (s), 158.7 (s), 138.8 (s),138.6 (s), 128.4, 128.2, 128.1, 127.7, 126.9, 126.2, 80.1 (d), 65.8 (d),62.6 (t), 27.7 (t), 20.4 (q).

Example 3 Benzhydryl 7-(dibromomethylene)cephalosporanate (4a)

To the solution of Ph₃P (12.0 g, 45.8 mmol) in anhydrous CH₂Cl₂ (75 mL)was added CBr₄ (7.6 g, 22.9 mmol) in one portion at 0° C. The mixturewas stirred at room temperature for 30 minutes. The reaction mixture wasthen cooled to −78° C. and a cold (−78° C.) solution of benzhydryl7-oxocephalosporanate 3 (5.00 g, 11.4 mmol) in anhydrous CH₂Cl₂ (40 mL)was added. After stirring at −78° C. for 30 minutes, the reaction wasconcentrated in vacuo and purified by column chromatography (CH₂Cl₂) togive a pale yellow solid (4.1 g, 61% yield). R_(f)=0.55 in CH₂Cl₂; mp58-60° C.; IR (CHCl₃) 3030, 1780, 1745 cm⁻¹; ¹H NMR (CDCl₃) δ 7.37 (10H,m), 6.96 (1H, s), 5.19 (1H, s), 4.97 (1H, d, A of ABq, J=13 Hz), 4.72(1H, d, B of ABq, J=13 Hz), 3.52 (1H, d, A of ABq, J=18 Hz), 3.32 (1H,d, A of ABq, J=18 Hz), 2.00 (3H, s); ¹³C NMR (CDCl₃) δ 170.2 (s), 160.5(s), 155.6 (s), 142.6 (s), 139.1 (s), 138.9 (s), 128.4, 128.0, 127.9,127.0, 126.7, 125.2 (s), 92.6 (s), 79.9 (d), 63.0 (t), 60.1 (d), 27.0(t), 20.5 (q). Anal. Calcd for C₂₄H₁₉NO₅SBr₂: C, 48.57; H, 3.20; N,2.36; Found: C, 48.52; H, 3.17; N, 2.19.

Example 4 Benzhydryl 7-(dichloromethylene)cephalosporanate (4b)

CCl₄ (2 mL, 20.7 mmol) was added into a solution of PPh₃ in anhydrousCH₃CN (50 mL) and stirred at room temperature for 30 minutes. Thissolution was transferred into a solution of benzhydryl7-oxocephalosporanate 3 (3.0 g, 8.9 mmol) in anhydrous CH₃CN (20 mL) andZn/Cu (1.0 g, 15 mmol) was added. This reaction mixture was furtherstirred at room temperature for 40 minutes. The unreacted Zn/Cu wasremoved by filtration and the filtrate was concentrated and purified bycolumn chromatography (CH₂Cl₂) to yield a pale yellow solid (2.70 g,78%). R_(f)=0.73 in CH₂Cl₂; mp 48-50° C.; IR (CHCl₃) 3050, 1780, 1740,940 cm⁻¹; ¹H NMR (CDCl₃) δ 7.38 (10H, m), 6.99 (1H, s) 5.29 (1H, s),5.02 (1H, d, A of ABq, J=13 Hz), 4.76 (1H, d, B of ABq, J=13 Hz), 3.57(1H, d, A of ABq, J=18 Hz) 3.88 (1H, d, B of ABq, J=18 Hz), 2.04 (3H,s); ¹³C NMR (CDCl₃) δ 170.0 (s), 160.2 (s), 154.5 (s), 138.8 (s), 138.7(s), 136.2 (s), 128.1, 127.7, 127.2, 126.6, 126.2, 124.7 (s), 123.6 (s),79.8 (d), 62.8 (t), 57.4 (d), 26.9 (t), 20.3 (q); high-resolution massspectrum for [C₂₄H₁₉NO₅SCl₂Na]⁺, i.e. [M+Na]⁺, m/z calcd 526.0259, found526.0251.

Example 5 Benzhydryl 7-[(E)-benzylidene]cephalosporanate and benzhydryl7-[(Z)-benzylidene]cephalosporanate (4c)

To a solution of triphenylbenzyl phosphonium bromide (11.44 g, 26.4mmol) in anhydrous THF (50 mL) was added a solution of n-BuLi (14.5 mL,29.0 mmol) at −78° C. The mixture was stirred at room temperature for 30minutes. The resulting red colored solution was recooled to −78° C. andwas added to a cold (−78° C.) solution of 7-oxocephalosporanate 3 (10.5g, 24.0 mmol) in anhydrous THF (25 mL) and stirred at −78° C. for 5minutes. The cold reaction mixture was then poured into ice coldsaturated NH₄Cl solution (25 mL) and the layers were separated. Theaqueous layer was extracted with ether (2×50 mL). The combined organiclayers were washed with water (25 mL), dried (Na₂SO₄), concentrated andpurified by column chromatography (CH₂Cl₂: Hexane, 3:1) to give theE-isomer (0.83 g, 40%), and the Z-isomer (1.26 g, 60%) as white fluffysolid.

7-(E)-isomer. R_(f)=0.60 in CH₂Cl₂; mp 59-61° C.; IR (CHCl₃) 3015, 1760,1730 cm⁻¹; ¹H NMR (CDCl₃) δ 7.83 (2H, m), 7.26 (13H, m), 6.93 (1H, s),6.53 (1H, s), 4.99 (1H, s), 4.78 (1H, d, A of ABq, J=13 Hz), 4.53 (1H,d, B of ABq, J=13 Hz), 3.39 (1H, d, A of ABq, J=18 Hz), 3.19 (1H, d, Bof ABq, J=18 Hz), 1.85 (3H, s); ¹³C NMR (CDCl₃) δ 170.2 (s), 161.1 (s),158.7 (s), 139.3 (s), 139.1 (s), 136.0 (s), 134.0 (d), 133.1, 130.3,128.6, 128.3, 128.0, 127.7, 127.0, 121.7 (s), 79.6 (d), 63.1 (t), 56.1(d), 27.9 (t), 20.5 (q). Anal. Calcd for C₃₀H₂₅NO₅S: C, 70.45; H, 4.89;N, 2.74. Found: C, 70.80; H, 5.03; N, 2.95.

7-(Z)-isomer. R_(f)=0.50 in CH₂Cl₂; mp 45-47° C.; IR (CHCl₃) 3025, 1790,1760 cm⁻¹; ¹H NMR (CDCl₃) δ 7.43 (15H, m), 7.21 (1H, d, J=1.18 Hz), 7.07(1H, s), 5.50 (1H, d, J=1.23 Hz), 5.00 (1H, d, A of ABq, J=13 Hz), 4.75(1H, d, B of ABq, J=13 Hz), 3.65 (1H, d, A of ABq, J=18 Hz), 3.41 (1H,d, B of ABq, J=18 Hz), 2.04 (3H, s); ¹³C NMR (CDCl₃) δ 170.3 (s), 161.0(s), 160.2 (s), 139.3 (s), 139.1 (s), 135.8(s), 132.4 (d), 130.5, 129.7,129.0, 128.3, 128.1, 127.9, 127.7, 127.0, 121.7(s), 79.7 (d), 63.1 (t),57.7 (d), 28.0 (t), 20.5 (q); high-resolution mass spectrum for[C₃₀H₂₅NO₅SNa]⁺, i.e. [M+Na]⁺, m/z calcd 534.1351, found 534.1352.

Example 6 Benzhydryl 7-[(Z)-(2′-pyridyl)methylene]cephalosporanate (4e)

To a solution of 2-picolyl chloride hydrochloride (13.1 g, 80 mmol) inwater (20 mL) was added into K₂CO₃ (11.0 g, 80 mmol). After thecarbonate was completely dissolved, the solution was extracted withether (3×10 mL). The combined organic layers were washed with saturatedNaCl solution (1×30 mL), dried (Na₂SO₄) and concentrated to give picolylchloride (9.2 g, 90%). Picolyl chloride (8.9 g, 70 mmol),triphenylphosphine (18.3 g, 70 mmol) and 1,4-dioxane (30 mL) were mixedand refluxed for 24 hours. The reaction mixture was washed with ether(2×30 mL) and the remaining solid was dried in vacuo to give a whitesolid (25.5 g, 94%). A mixture of 2-picolyltriphenylphosphonium chloride(5.8 g, 15 mmol) and sodium amide (0.58, 15 mmol) in THF (15 mL) wasstirred at room temperature for 30 minutes. The resulting brownsuspension was cooled to −78° C. and a solution of benzhydryl7-oxocephalosporanate 3 (6.6 g, 15 mmol) in THF (15 mL) was added in oneportion and the mixture was stirred at −78° C. for 15 minutes. Thereaction was quenched by the addition of saturated ammonium chloridesolution (10 mL) and the reaction mixture extracted with EtOAc (2×20mL). The combined organic layers were washed with water (2×40 mL), driedover MgSO₄, concentrated and purified by column chromatography to obtaina yellow solid (2.9 g, 38%). R_(f)=0.28 in 2% EtOAc in CH₂Cl₂; mp181-183° C.; IR (CHCl₃) 3060, 1810, 1750 cm⁻¹; ¹H NMR (CDCl₃) δ 8.68(1H, d), 7.72 (1H, t), 7.35(12H, m), 7.15 (1H, s), 7.10 (1H, s), 5.66(1H, s), 4.96 (1H, d, A of ABq, 13 Hz), 4.73 (1H, d, B of ABq, J=13 Hz),3.63 (1H, d, A of ABq, J=18 Hz), 3.63 (1H, D, B of ABq, J=18 Hz), 2.01(3H, s); ¹³C NMR (CDCl₃) δ 170.3 (s), 161.0 (s), 160.2 (s), 151.6 (d),150.1 (s), 140.6 (s), 139.3 (s), 139.1 (s), 136.6 (d), 128.3, 127.9,127.8, 127.6, 127.2, 126.9, 125.8 (s), 123.9 (s), 123.5 (s), 79.5 (d),63.0 (t), 58.5 (d), 28.0 (t), 20.5 (q); high-resolution mass spectrumfor [C₂₉H₂₄N₂O₅SNa]⁺, i.e. [M+Na]⁺, m/z calcd 535.1304, found 535.1300.

Example 7 Benzhydryl 7-[(Z)-methoxycarbonylmethylene]cephalosporanate(4f)

To a solution of benzhydryl 7-oxocephalosporanate (1.0 g, 2.3 mmol) inanhydrous CH₂Cl₂ (20 mL) at −78° C. was addedmethyl(triphenylphosphoranylidene)acetate (0.67 g, 2.0 mmol). Themixture was then stirred at −78° C. for 30 minutes. Acetic acid (0.5 mL)was added to quench the reaction and the reaction mixture wasconcentrated and purified by column chromatography to give titlecompound as a pale yellow solid (0.67 g, 68%). R_(f)=0.42 in 2% EtOAc inCH₂Cl₂; mp 47-49° C.; IR (CHCl₃) 3050, 1790, 1730 cm⁻¹; ¹H NMR (CDCl₃) δ7.36 (10H, m), 7.00 (1H, s), 6.49 (1H, s), 5.50 (1H, s), 5.00 (1H, d, Aof ABq, J=13.48 Hz), 4.76 (1H, d, B of ABq, J=13.47 Hz), 3.84 (3H, s),3.64 (1H, d, A of ABq, J=18.75 Hz), 3.39 (1H, d, B of ABq, J=18.75 Hz),2.03 (3H, s); ¹³C NMR (CDCl₃) δ 170.3 (s), 163.8 (s), 160.6 (s), 157.5(s), 152.6 (s), 139.2 (s), 139.0 (s), 128.6, 128.3, 127.9, 127.6, 127.3,126.90, 125.45 (s), 117.51 (d), 79.90 (d), 62.96 (t), 57.88 (d), 52.48(q), 27.91 (t). 20.59 (q). Anal. Calcd for C₂₆H₂₃NO₇S: C, 63.29; H,4.66; N, 2.84. Found: C, 63.47; H, 4.73; N. 2.87.

Example 8 Benzhydryl 7-[(Z)-t-butoxycarbonylmethylene]cephalosporanate(4g)

To a solution of benzhydryl 7-oxocephalosporanate in example 2 (4.0 g,9.2 mmol) in anhydrous CH₂Cl₂ (40 mL) at −78° C. was added a solution oft-butyl(triphenyl-phosphoranylidene)acetate (3.45 g, 9.15 mmol in 40 mLCH₂Cl₂). The mixture was then stirred at −78° C. for 30 minutes. Aceticacid (1 mL) was added to quench the reaction and the reaction mixturewas concentrated and purified by column chromatography to give titlecompound as a pale yellow solid (yield=55%). R_(f)=0.52 in 2% EtOAc inCH₂Cl₂. mp 48-50° C.; IR (CHCl₃) 3050, 1780, 1730 cm⁻¹; ¹H NMR (CDCl₃) δ7.36 (10H, m), 7.00 (1H, s), 6.39 (1H, s), 5.47 (1H, s), 5.00 (1H, d, Aof ABq, J=13.48 Hz), 4.77 (1H, d, B of Abq, J=13.48 Hz), 3.62 (1H, d, Aof ABq, J=18 Hz), 3.38 (1H, d, B of ABq, J=18 Hz), 2.02 (3H, s), 1.54(9H, s); ¹³C NMR (CDCl₃) δ 170.2 (s), 162.4 (s), 160.5 (s), 157.8 (s),150.1, (s), 139.0 (s), 138.8 (s), 128.3, 128.0, 127.9, 127.5, 126.9,125.0 (s), 119.9 (d), 82.9 (s), 79.7 (d), 62.8 (t), 57.5 (d), 28.0 (q),27.9 (t), 20.4 (q). Anal. Calcd for C₂₉H₂₉NO₇S: C, 65.05; H, 5.42; N,2.62. Found: C, 64.50; H, 5.42, N, 2.62.

Example 9 Benzhydryl 7-[(Z)-acetylmiaphylene]cephalosporanate (4h)

This compound was prepared as described for compound example 8(yield=58%). R_(f)=0.29 in 2% EtOAC in CH₂Cl₂; mp 49-50° C.; IR (CHCl₃)3000, 1770, 1720 cm⁻¹; ¹H NMR (CDCl₃) δ 7.36 (10H, m), 7.00 (1H, s),6.48 (1H, s), 5.50 (1H, s), 5.00 (1H, d, A of ABq, J=13.47 Hz), 4.77(1H, d, B of ABq, J=13.48 Hz), 3.63 (1H, d, A of ABq, J=18.75 Hz), 3.38(1H, d, B of ABq, J=18.75 Hz). 2.39 (3H, s), 2.02 (3H, s). ¹³C NMR(CDCl₃) δ 195.8 (s), 170.3 (s), 160.6 (s), 158.5 (s), 149.5 (s), 139.3(s), 139.1 (s), 128.5, 127.8, 127.1, 126.9, 126.3, 125.6 (s), 122.7 (d),79.8 (d), 63.0 (t), 58.0 (d), 30.9 (q), 28.0 (t), 20.7 (q). Anal. Calcdfor C₂₆H₂₃NO₆S: 65.41, 4.82, 2.94. Found: C, 65.89; H, 4.87; N, 3.11.

Example 10 Benzhydryl 7-[(Z)-formylmethylene]cephalosporanate (4i)

This compound was prepared as described for compound example 8(yield=46%). R_(f)=0.37 in 2% EtOAc in CH₂Cl₂; mp 113-115° C.; IR(CHCl₃) 3050, 1780. 1730, 1700 cm⁻¹; ¹H NMR (CDCl₃) δ 7.39 (10H, m),6.99 (1H, s), 6.60 (1H, d, J=6.17 Hz), 5.45 (1H, s), 5.00 (1H, d, A ofABq, J=13.51 Hz), 4.75 (1H, d, B of ABq, 13.55 Hz), 3.64 (1H, d, A ofABq, J=18.59 Hz), 3.41 (1H, d, B of ABq, J=18.61 Hz), 2.00 (3H, s); ¹³CNMR (CDCl₃) δ 188.2 (d), 170.1 (s), 160.3 (s), 157.0 (s), 154.7 (s),138.9 (s), 138.8 (s), 128.4, 128.1, 128.0, 127.6, 126.9, 126.7, 125.0(s), 123.5 (d), 79.9 (d), 62.4 (t), 56.4 (d), 28.1 (t), 20.4 (q);high-resolution mass spectrum for [C₂₅H₂₁NO₆SNa]⁺, i.e. [M+Na]⁺, m/zcalcd 486.0987, found 468.0981.

Example 11 Benzhydryl 7-[(Z)-hydroxymethylmethylene]cephalosporanate(4j)

To a solution of the formylmethylene cephalosporanate described inexample 10 (0.75 g, 1.62 mmol) in methanol (10 mL) and acetic acid (1mL) was added NaCNBH₃ (0.51 g, 8.1 mmol) in one portion, and stirred atroom temperature for 30 minutes. The reaction mixture was concentratedin vacuo, and the residue was dissolved in EtOAc (25 mL) and water (10mL). The aqueous layer was exacted with EtOAc (1×30 mL), and thecombined organic layer was washed with water (1×30 mL), dried (Na₂SO₄),concentrated and purified by column chromatography to give a white solid(0.71 g, 94%). R_(f)=0.3 in 10% EtOAc in CH₂Cl₂; mp 58-60° C.; ¹H NMR(CDCl₃) δ 7.39 (10H, s), 7.01 (1H, s), 6.51 (1H, s), 5.29 (1H, s), 4.94(1H, d, A of ABq, J=13.16 Hz), 4.71 (1H, d, B of ABq, J=13.18 Hz), 4.60(1H, d, A of ABq, J=20.83 Hz), 4.42 (1H, d, B of ABq, J=20.22 Hz), 3.56(1H, d, A of ABq, J=18.37 Hz), 3.33 (1H, d, B of ABq, J=18.09 Hz), 2.01(3H, s); ¹³C NMR (CDCl₃) δ 170.5 (s), 161.2 (s), 159.9 (s), 139.0 (s),138.8 (s), 137.4 (s), 131.8 (d), 128.3, 128.0, 127.9, 127.6, 127.4,126.8, 122.2 (s), 79.6 (d), 63.0 (t), 60.0 (t), 56.9 (d), 28.0 (t), 20.5(q); high-resolution mass spectrum for [C₂₅H₂₃NO₆SNa]⁺, i.e. [M+Na]⁺,m/z calcd 488.1144, found 488.1138.

Example 12 Benzhydryl7-[(Z)-N-methoxy-N-methylaminocarbonylmethylene]cephalosporanate (4k)

To a solution of benzhydryl 7-oxocepharosporanate (1.0 g, 2.3 mmol) inanhydrous CH₂Cl₂ (20 mL) at −78° C. was addedN-methoxy-N-methyl-2-(triphenyl-phosphoranylidene)acetamide (0.73 g, 2.0mmol). The mixture was stirred at −78° C. for 10 minutes, and thenwarmed to 0° C. and further stirred for 15 minutes. Acetic acid (0.5 mL)was added to quench the reaction, and the reaction mixture wasconcentrated and purified by column chromatography (2% EtOAc in CH₂Cl₂)to give title compound as a pale yellow solid (0.53 g, 51%). IR (CHCl₃)3050, 1780, 1730 cm⁻¹; ¹H NMR (CDCl₃) δ 7.35 (10H, m), 7.06 (1H, s),7.00 (1H, s), 5.56 (1H, s), 4.96 (1H, d, A of ABq, J=13.40 Hz), 4.75(1H, d, B of ABq, J=13.52 Hz), 3.75 (3H, s), 3.64 (1H, d, B of ABq,J=18.43 Hz), 3.37 (1H, d, B of ABq, J=18.97 Hz), 3.28 (3H, s), 2.01 (3H,s); ¹³C NMR (CDCl₃) δ 170.4 (s), 163.1 (s), 160.8 (s), 158.5 (s), 151.2(s), 139.2 (s), 139.0 (s), 128.5, 128.4, 128.1, 128.0, 127.8, 127.0,124.8 (s), 115.6 (d), 79.8 (d), 63.0 (t), 62.4 (q), 58.0 (d), 32.2 (q),28.1 (t), 20.6 (q).

Example 13 Benzhydryl 7-[(E)-bromomethylene]cephalosporanate (41)

To a solution of 7-(dibromomethylene)cephalosporanate (1.19 g, 2 mmol)in methanol (20 mL) and THF (10 mL) was added NH₄Cl (8.56 g, 16 mmol) inone portion at 0° C. The mixture was stirred for 5 minutes. Zn/Cu (5.20g, 8 mmol) was added in one portion and further stirred at roomtemperature for 30 minutes. The solvent was removed, and residue wasextracted with ether (2×20 mL). The obtained ether was washed with water(1×20 mL) and brine (1×10 mL), dried (Na₂SO₄), concentrated and purifiedby column chromatography (CH₂Cl₂) to give a white solid (0.86 g, 83%yield). R_(f)=0.41 in CH₂Cl₂; mp 48-50° C.; IR (CHCl₃) 3025, 1780, 1730cm⁻¹; ¹H NMR (CDCl₃) δ 7.32 (10H, m), 6.96 (1H, s), 6.44 (1H, s), 5.05(1H, s) 4.92 (1H, d, A of ABq, J=13.37 Hz), 4.67 (1H, d, B of ABq,J=13.36 Hz), 3.46 (1H, d, A of ABq, J=18.31 Hz), 3.26 (1H, d, B of ABq,J=18.37 Hz), 1.96 (3H, s); ¹³C NMR (CDCl₃) δ 170.15 (s), 160.60 (s),157.04 (s), 141.77 (s), 139.05 (s), 138.86 (s), 128.32, 127.97, 127.89,127,49, 126.92, 123.30 (s), 107.94 (d), 79.82 (d), 62.90 (t), 58,02 (d),27.68 (t), 20.42 (q). Anal. Calcd for C₂₄H₂₀NO₅SBr: C, 56.03; H, 3.89;N, 2.72. Found: C, 56.29; H, 3.87; N, 2.63.

Example 14 Benzhydryl 7-[(Z)-t-butylmethylene]cephalosporanate (4m)

To a suspension of CuCN (1.65 g, 3.2 mmol) in anhydrous THF (50 mL) at−78° C. was added t-BuLi (3.8 mL, 4.2 mmol). The cooling bath wasremoved until all the solid had gone into the solution. This cupratesolution was cooled to −78° C. and a solution of benzhydryl7-(E)-bromomethylene cephalosporanate (1.65 g, 3.2 mmol in anhydrousTHF, 15 mL) at −78° C. was cannulated to the cuprate solution as fast aspossible. The solution was stirred at −78° C. for 1 minutes beforequenching with saturated NH₄Cl solution (20 mL). The reaction mixturewas extracted with ether (50 mL). The combined organic layers werewashed with cold saturated NH₄Cl (2×10 mL), dried aver Na₂SO₄,concentrated, and purified by column chromatography (CH₂Cl₂) to give awhite solid (1.23 g, 78% yield). R_(f)=0.64 in CH₂Cl₂; mp 120-121° C.;IR (CHCl₃) 2950, 1765, 1730 cm⁻¹; ¹H NMR (CDCl₃) δ 7.35 (10H, m), 7.00(1H, s), 6.00 (1H, s), 4.93 (1H, s), 4.86 (1H, d, A of ABq, J=13.07 Hz),4.63 (1H, d, B of ABq, J=13.05 Hz), 3.48 (1H, d, A of ABq, J=18.30 Hz),3.28 (1H, d, B of ABq, J=18.32 Hz), 1.96 (3H, s), 1.24 (9H, s); ¹³C NMR(CDCl₃) δ 170.30 (s), 161.44 (s), 158.31 (s), 147.87 (d), 139,30 (s),139.08 (s), 135.76 (s), 128.31, 128.02, 127.80, 127.03, 121.01 (s),79,50 (d), 63.10 (t), 55.72 (d), 34.43 (s), 29.83 (q), 27.86 (t), 20.50(q). Anal. Calcd for C₂₈H₂₉NO₅S: C, 68.43; H, 5.91; N, 2.85. Found: C,67.98; H, 5.90; N, 2.72.

Example 15 Benzhydryl 7-[dibromomethylene]cephalosporanate Sulfone (5a)

To a solution of the corrsponding sulfide 4a (0.3 g, 0.5 mmol) in CH₂Cl₂(10 mL) and pH=6.4 Buffer solution (10 mL) was added m-CPBA (85%, 0.35g, 2.0 mmol) in one portion. The mixture was stirred at room temperaturefor 40 minutes, and then ether (50 mL) was added. After separatinglayers, the organic layers were washed with saturated NaHCO₃ (3×30 mL),dried (NaSO₄), concentrated and purified by column chromatography toyield a white solid (2.5 g, 79%). R_(f)=0.50 in 2% EtOAc in CH₂Cl₂; mp62-64° C.; IR (CHCl₃) 3030, 1800, 1740, 1350, 1130 cm⁻¹; ¹H NMR (CDCl₃)δ 7.36 (10H, m), 6.95 (1H, s), 5.20 (1H, s), 5.03 (1H, d, A of ABq,J=14.18 Hz), 4.68 (1H, d, B of ABq, J=14.16 Hz), 4.02 (1H, d, A of ABq,J=18.38 Hz), 3.77 (1H, d, B of ABq, J=18.40 Hz), 2.02 (3H, s); ¹³C NMR(CDCl₃) δ 170.1 (s) , 159.6 (s), 154.8 (s), 138.8 (s), 138.7 (s), 135.2(s), 128.6, 128.3, 127.5, 127.1, 126.4, 125.5 (s), 124.1(s), 98.2 (s),80.8 (d), 73.0 (d), 62.0 (t), 52.1 (t), 20.5 (q). Anal. Calcd forC₂₄H₁₉NO₇SBr₂: C,46.08; H, 3.04; N, 2.24; Br, 25.60. Found: C, 46.29; H,3.09; N, 2.13, Br, 26.18.

Example 16 Benzhydryl 7-[dichloromethylene]cephalosporanate Sulfone (5b)

This compound was prepared form the corresponding sulfide 4b asdescribed for the compound in example 15 to give a white solid(yield=81%). R_(f)=0.38 in 2% EtOAc in CH₂Cl₂; mp 64-66° C.; IR (CHCl₃)3050, 1800, 1740, 1350, 1140 cm⁻¹; ¹H NMR (CDCl₃) δ 7.35 (10H, m), 6.95(1H, s), 5.28 (1H, s), 5.05 (1H, d, A of ABq, 14.14 Hz), 4.65 (1H, d, Bof ABq, 13.90 Hz), 4.03 (1H, d, A of ABq, J=18.08 Hz), 3.80 (1H, B ofABq, 17.74 Hz), 2.04 (3H, s); ¹³C NMR (CDCl₃) δ 170.2 (s), 159.6 (s),153.9 (s), 138.6 (s), 138.5 (s), 134.3 (s), 130.2 (s), 128.9, 128.6,128.3, 127.6, 127.3, 127.1, 124.3 (s), 80.7 (d), 70.7 (d), 61.9 (t),51.7 (t), 20.5 (q). Anal. Calcd for C₂₄H₁₉NO₇SCl: C, 53.73; H, 3.54; N,2.61. Found: C, 53.36; H, 3.78; N, 2.47.

Example 17 Benzhydryl 7-[(E)-benzylidene]cephalosporanate Sulfone (5c)

This compound was prepared from the sulfide 4c (0.51 g, 1.0 mmol) asdescribed for the compound in example 15 to give a white solid (0.350 g,yield=65%). R_(f)=0.27 in CH₂Cl₂. mp 194-196° C.; IR (CHCl₃) 2975, 1775,1730, 1340, 1125 cm⁻¹; ¹H NMR (CDCl₃) δ 8.00 (2H, m), 7.41 (13H, m),7.03 (1H, s), 6.94 (1H, s), 5.24 (1H, s), 5.04 (1H, d, A of ABq, J=13.91Hz), 4.70 (1H, d, B of ABq, J=13.98 Hz), 4.05 (1H, d, A of ABq, J=17.96Hz), 3.77 (1H, d, B of ABq, J=18.13 Hz), 2.05 (3H, s). ¹³C NMR (CDCl₃) δ170.3 (s), 160.1(s), 157.7 (s), 138.9 (s), 138.8 (s), 138.5(d), 132.5,131.5, 131.0, 128.9, 128.6, 128.3, 127.7, 127.1, 126.7, 122.8 (s), 80.4(d), 69.5 (d), 62.1 (t), 51.2 (t), 20.5 (q); high-resolution massspectrum for [C₃₀H₂₅NO₇SNa]⁺, i.e. [M+Na]⁺, m/z calcd 566.1249, found566.1248.

Example 18 Benzhydryl 7-[(Z)-benzylidene]cephalosporanate Sulfone (5d)

This compound was prepared from the sulfide 4d (0.68 g, 1.3 mmol) asdescribed for the compound in example 15 to give a white solid(yield=57%, 0.410 g). R_(f)=0.40 in CH₂Cl₂. MP 61-63° C. IR (CHCl₃)3025, 2925, 1780, 1730, 1340, 1130 cm⁻¹; ¹H NMR (CDCl₃) δ 7.42 (15H, m),7.12 (1H, s), 6.98 (1H, s), 5.53 (1H, s), 4.95 (1H, d, A of ABq, J=13.80Hz), 4.65 (1H, d, B of ABq, J=13.92 Hz), 4.04 (1H, d, A of ABq, J=18.33Hz), 3.77 (1H, d, B of ABq, J=18.50 Hz), 1.96 (3H, s); ¹³C NMR (CDCl₃) δ170.1 (s), 159.9 (s), 159.7 (s), 138.8 (s), 138.7 (s), 134.12 (s), 131.6(d), 131.0, 129.8, 129.1, 128.4, 128.2, 128.1, 127.6, 127.0, 126.7,126.2, 121.8 (d), 80.3 (d), 71.7 (d), 691.9 (t), 51.6 (t), 20.3 (q);high-resolution mass spectrum for [C₃₀H₂₅NO₇SNa]⁺, i.e. [M+Na]⁺, m/zcalcd 566.1249, found 566.1262.

Example 19 Benzhydryl 7-[(Z)-(2′-pyridyl)methylene]cephalosporanateSulfone (5e)

This compound was prepared from the corresponding sulfide 4e (0.45 g,0.88 mmol) as described for the compound in example 15 to give a whitesolid (yield=90%). R_(f)=0.26 in 2% EtOAc in CH₂Cl₂; mp 120-1.22° C.; IR(CHCl₃) 2975, 2950, 1780, 1720, 1340, 1130 cm⁻¹; ¹H NMR (CDCl₃) δ 8.67(1H, d), 7.71 (1H, t), 7.40 (13H, m), 7.00 (1H, s), 5.91 (1H, s), 5.14(1H, d, A of ABq, J=14.07 Hz), 4.80 (1H, B of ABq, J=14.06 Hz), 4.11(1H, d, A of ABq, J=17.58 Hz), 3.78 (1H, d, B of ABq, J=17.58 Hz), 2.05(3H, s); high-resolution mass spectrum for [C₂₉H₂₄N₂O₇SNa]⁺, i.e.[M+Na]⁺, m/z calcd 567.1202, found 567.1198.

Example 20 Benzhydryl 7-[(Z)-t-butoxycarbonylmethylene]cephlosporanateSulfone (5g)

This compound was prepared from the corresponding sulfide 4g asdescribed for the compound in example 15 to give a white solid(yield=73%). R_(f)=0.68 in 5% EtOAc in CH₂Cl₂; mp 58-60° C. IR (CHCl₃)3025, 1800, 1730, 1350, 1160 cm⁻¹; ¹H NMR (CDCl₃) δ 7.36 (10H, m), 6.98(1H, s), 6.59 (1H, s), 5.58 (1H, s), 5.14 (1H, d, A of ABq, J=14.35 Hz),4.80 (1H, d, B of ABq, J=14.35 Hz), 4.12 (1H, d, A of ABq, J=17.58 Hz),3.77 (1H, d, B of ABq, J=17.87 Hz), 2.04 (3H, s). 1.52 (9H, s); ¹³C NMR(CDCl₃) δ 170.0 (s), 161.5 (s), 159.4 (s), 157.1 (s), 142.3 (s), 138.6(s), 138.5 (s), 128.8, 128.4, 128.3, 127.2, 127.0, 125.9 (s), 123.5 (d),83.8 (s), 80.2 (d), 71.6 (d), 61.3 (t), 52.8 (t), 27.6 (q), 20.2 (q);high-resolution mass spectrum for [C₂₉H₂₉NO₉SNa]⁺, i.e. [M+Na]⁺, m/zcalcd 590.1461, found 590.1447.

Example 21 Benzhydryl 7-[(Z)-acetylmethylene]cephlosporanate Sulfone(5h)

This compound was prepared from the corresponding sulfide 4h asdescribed for the compound in example 15 to give a white solid(yield=79%). R_(f)=0.66 in 25% EtOAc in CH₂Cl₂; mp 176-178° C.; IR(CHCl₃) 3050, 1800, 1730, 1350, 1140 cm⁻¹; ¹H NMR (CDCl₃) δ 7.38 (10H,m), 6.99 (1H, s), 6.94 (1H, s), 5.64 (1H, s), 5.13 (1H, d, A of ABq,J=14.51 Hz), 4.81 (1H, d, B of ABq, J=14.41 Hz), 4.12 (1H, d, A of ABq,J=17.91 Hz), 3.80 (1H, d, B of ABq, J=17.94 Hz), 2.46 (3H, s), 2.07 (3H,s). ¹³C NMR (CDCl₃) δ 194.7 (s), 170.1 (s), 159.5 (s), 157.5 (s), 141.2(s), 138.7 (s), 138.6 (s), 128.6, 128.3, 127.5, 127.1, 126.8 (s), 125.3(d), 80.5 (d), 72.2 (d), 61.7 (t), 53.1 (t), 31.0 (q), 20.5 (q);high-resolution mass spectrum for [C₂₆H₂₃NO₈SNa]⁺, i.e. [M+Na]⁺, m/zcalcd 532.1042, found 532.1045.

Example 22 Benzhydryl 7-[(Z)-hydroxymethylmethylene]cephalosporanateSulfone (5j)

This compound was prepared form the corresponding sulfide 4j asdescribed in the compound in example 15 to give a white solid(yield=68%). R_(f)=0.32 in 25% EtOAc in CH₂Cl₂; mp 58-60° C.; IR (CHCl₃)3050, 1780, 1730, 1330, 1130 cm⁻¹; ¹H NMR (CDCl₃) δ 7.36 (10H, m), 6.97(1H, s), 6.81 (1H, s), 5.53 (1H, s), 5.05 (1H, d, A of ABq, J=14.06 Hz),4.74 (1H, d, B of ABq, J=14.06 Hz), 4.61 (1H, d, A of ABq, J=19.04 Hz),3.98 (1H, d, B of ABq, J=19.03 Hz), 4.06 (1H, d, A of ABq, J=17.0 Hz),3.76 (1H, d, B of ABq, J=17.80 Hz), 2.04 (3H, s); ¹³C NMR (CDCl₃) δ170.4 (s), 160.0 (s), 158.6 (s), 138.9 (s), 138.8 (s), 136.8 (d), 128.6,128.1, 127.8, 127.6, 127.5, 127.2, 126.7, 124.3 (s), 80.8 (d), 71.4 (d),61.9 (t), 60.7 (t), 51.4 (t), 20.6 (q); high-resolution mass spectrumfor [C₂₅H₂₃NO₆SNa]⁺, i.e. [M+Na]⁺, m/z calcd 488.1144, found 488.1138.

Example 23 Benzhydryl7-[(Z)-N-methoxy-N-methylaminocarbonylmethylene]cephlosporanate Sulfone(5k)

This compound was prepared from the corresponding sulfide 4k asdescribed for the compound in example 15 to give a white solid(yield=68%). R_(f)=0.44 in 25% EtOAc in CH₂Cl₂; mp 81-82° C.; IR (CHCl₃)3050, 1800, 1740, 1360, 1140 cm⁻¹; ¹H NMR (CDCl₃) δ 7.36 (10H, m), 7.28(1H, s), 6.98 (1H, s), 5.72 (1H, s), 5.10 (1H, d, A of ABq, J=14.06 Hz),4.82 (1H, d, B of ABq, J=14.35 Hz), 4.11 (1H, d, A of ABq, J=16.70 Hz),3.78 (1H, d, B of ABq, J=17.58 Hz), 3.78 (3H, s), 3.31 (3H, s), 2.06(3H, s); ¹³C NMR (CDCl₃) δ 170.1 (s), 162.1 (s), 159.7 (s), 157.8 (s),142.78 (s), 138.9 (s), 138.8 (s), 128.7, 128.4, 127.7, 127.4, 127.1,126.9, 125.7 (s), 119.3 (d), 80.3 (d), 72.3 (d), 62.5 (q), 61.8 (t),52.9 (t), 32.3 (q), 20.5 (q); high-resolution mass spectrum for[C₂₇H₂₆N₂O₉SNa]⁺, i.e. [M+Na]⁺, m/z calcd 577.1257, found 577.1247.

Example 24 Benzhydryl 7-[(E)-bromomethylene]cephalosporanate Sulfone(5l)

This compound was prepared from the corresponding sulfide 4l asdescribed for the compound in example 15 to give a white solid(yield=71%). R_(f)=0.43 in 2% EtOAC in CH₂Cl₂; mp 80-82° C.; IR (CHCl₃)3030, 1800, 1730, 1350, 1130 cm⁻¹; ¹H NMR (CDCl₃) δ 7.33 (10H, m), 6.94(1H, s), 6.91 (1H, s), 5.10 (1H, s), 5.00 (1H, d, A of ABq, J=14.19 Hz),4.67 (1H, d, B of ABq, J=14.17 Hz), 3.97 (1H, A of ABq, J=18.06 Hz),3.69 (1H, d, B of ABq, J=18.17 Hz), 1.99 (1H, s); ¹³C NMR (CDCl₃) δ170.1 (s), 159.7 (s), 156.3 (s), 138.7 (s), 138.6 (s), 134.0 (s), 128.4,128.1, 127.3, 126.9, 125.7, 124.9 (s), 112.5 (d), 80.57 (d), 70.9 (d),61.8 (t), 51.2 (t), 20.4 (q). Anal. Calcd for C₂₄H₂₀NO₇SBr: C, 52.75; H,3.66; N, 2.56. Found: C, 52.78, H, 3.75; N, 2.77.

Example 25 Benzhydryl 7-[(Z)-t-butylmethylene]cephalosporanate Sulfone(5m)

This compound was prepared from the corresponding sulfide 4m asdescribed in the compound in example 15 to give a white solid(yield=84%). R_(f)=0.48 in 2% EtOAc in CH₂Cl₂; mp 147-149° C.; IR(CHCl₃) 3050, 3000, 1785, 1740, 1340, 1130 cm⁻¹; ¹H NMR (CDCl₃) δ 7.37(10H, m), 6.97 (1H, s), 6.26 (1H, d, J=1.0 Hz), 5.01 (1H, d, J=1.0 Hz),4.97 (1H, d, A of ABq, J=13.86 Hz), 4.65 (1H, d, B of ABq, J=13.83 Hz),3.96 (1H, d, A of ABq, J=18.27 Hz), 3.82 (1H, d, B of ABq, J=17.95),2.01 (3H, s), 1.29 (9H, s); ¹³C NMR (d⁶-acetone) δ 170.40 (s), 160.92(s), 158.49 (s), 152.29 (d), 140.44 (s), 140.24 (s), 128.89, 128.63,128.00, 127.58, 126.43, 124.24 (s), 80.42 (d), 69.70 (d), 62.41 (t),51.15 (t), 35.40 (s), 29.67 (q), 20.33 (q); high-resolution massspectrum for [C₂₈H₂₉NO₇SNa]⁺, i.e. [M+Na]⁺, m/z calcd 546.1562, found546.1551.

Example 26 Sodium Salt of 7-[dibromomethylene]cephalosporanic Acid (6a)

To a solution of benzhydryl 7-dibromomethylene cephalosporanate 4a (0.3g, 0.5 mmol) in anhydrous CH₂Cl₂ (10 mL) was added anisole (0.54 mL, 5mmol) at −78° C. followed by the addition of AlCl₃ solution (1.25 mL,1.25 mmol) in one portion. The mixture was stirred at −78° C. for 15minutes and poured into rapidly stirred cold water (30 mL) containingNaHCO₃ (0.42 g, 5 mmol), followed by the addition of EtOAc (30 mL). Itwas further stirred for 5 minutes and filtered using celite 545. Theaqueous layer was separated and concentrated in vacuo to about 2 mL andpurified by reverse phase chromatography followed by lyophilization toyield a pink solid (180 mg, 80%). R_(f)=0.62 in 10% EtOH in water;UV:λ_(max)=252 nm (50 mM phosphate buffer, pH=7.2), ε=14,434 cm⁻¹.mol⁻¹. l; IR (KBr) 2950, 1730, 1600, 1390 cm⁻¹; ¹H NMR (d⁶-DMSO) δ 5.42(1H, s), 4.91 (1H, d, A of ABq, J=12.15), 4.71 (1H, d, B of ABq, J=11.89Hz), 3.50 (1H, d, A of ABq, J=17.39 Hz), 3.22 (1H, d, B of ABq, J=17.72Hz), 1.99 (3H, s); high-resolution mass spectrum for [C₁₁H₈NO₅SBr₂Na₂]⁺,i.e. [M+Na]⁺, m/z calcd 469.8285, found 469.8277.

Example 27 Sodium Salt of 7-[dichloromethylene]cephalosporanic Acid (6b)

This compound was prepared from the corresponding ester 4b (0.3 g, 0.6mmol) as described in the compound in example 26 to give a pale yellowfluffy solid (yield=62%). R_(f)=0.66 in 10% EtOH in water;UV:λ_(max)=242 nm (50 mM phosphate buffer, pH=7.2), ε=11,789cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1740, 1600, 1390 cm⁻¹; ¹H NMR (d⁶-DMSO)ε5.52 (1H, s), 4.93 (1H, d, A of ABq, J=12.09 Hz), 4.73 (1H, d, B ofABq, J=12.70 Hz), 3.53 (1H, d, A of ABq, J=17.65 Hz), 3.27 (1H, d, B ofABq, J=17.62 Hz), 1.99 (3H, s); high-resolution mass spectrum for[C₁₁H₉NO₅SCl₂Na]⁺, i.e. [M+H]⁺, m/z calcd 359.9473, found 359.9476.

Example 28 Sodium Salt of 7-[(Z)-(2′-pyridyl)methylene]cephalosporanicAcid (6c)

This compound was prepared from the corresponding ester 4c (0.4 g, 0.78mmol) as described for the compound in example 26 to give a yellow solid(149 mg, 52%). R_(f)=0.56 in 10% EtOH in water; UV:λ_(max)=296 nm (50 mMphosphate buffer, pH=7.2), ε=11,257 cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1720,1600, 1390 cm⁻¹; ¹H NMR (d⁶-DMSO) δ 8.64 (1H, d), 7.83 (1H, t), 7.63(1H, d), 7.37 (1H, t), 7.34 (1H, s), 5.63 (1H, s), 4.92 (1H, d, A ofABq, J=11.43 Hz), 4.77 (1H, d, B of ABq, J=12.30 Hz), 3.56 (1H, d, A ofABq, J=17.57 Hz), 3.27 (1H, d, B of ABq, J=17.78 Hz), 2.00 (3H, s);high-resolution mass spectrum for [C₁₆H₁₄N₂O₅SNa]⁺, i.e. [M+H]⁺, m/zcalcd 369.0518, found 369.0506.

Example 29 Sodium Salt of 7-[(Z)-methoxycarbonylmethylene]cephlosporanicAcid (6f)

To a solution of benzhydryl 7-(Z)-(methoxycarbonylmethylene)cephalosporanate 4f (0.25 g, 0.51 mmol) in anisole (1.1 mL, 15.3 mmol)at 0° C. was added trifluroacetic acid (4.6 mL, 59.7 mmol). The mixturewas stirred for 10 minutes, concentrated in vacuo, dissolved in 40 mLEtOAc, and then poured into rapidly stirred NaHCO₃ solution (0.5 g in 30mL H₂O). The aqueous layer was separated, concentrated in vacuo to 2 mLand further purified by reverse phase chromatography (R_(f)=0.84 in 5%EtOH in water) followed by lyopholization to yield a pale yellow fluffysolid (158 mg, 89%). UV:λ_(max)=226 nm (50 mM phosphate buffer, pH=7.2),ε=12,254 cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1730, 1600, 1400 cm⁻¹; ¹H NMR(d⁶-DMSO) δ 6.47 (1H, s), 5.54 (1H, s), 4.93 (1H, d, A of ABq, J=11.71Hz), 4.79 (1H, d, B of ABq, J=11.72 Hz), 3.37 (3H, s), 3.58 (1H, d, A ofABq, J=18.31 Hz), 3.27 (1H, d, B of ABq, J=17.58 Hz), 2.01 (3H, s);high-resolution mass spectrum for [C₁₃H₁₃NO₇SNa]⁺, i.e. [M+H]⁺, m/zcalcd 350.0310, found 350.0310.

Example 30 Sodium Salt of7-[(Z)-t-butoxycarbonylmethylene]cephlosporanic Acid (6g)

This compound was prepared from the corresponding ester 4g (0.3 g, 0.56mmol) as described for example 29 to give a yellow fluffy solid (176 mg,81%). R_(f)=0.53 in 5% EtOH in water; UV:λ_(max)=225 nm (50 mM phosphatebuffer, pH=7.2), ε=11,324 cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1720, 1600, 1400cm⁻¹; ¹H NMR (d⁶-DMSO) δ 6.26 (1H, s), 5.47 (1H, s), 4.92 (1H, d, A ofABq, J=12.00 Hz), 4.72 (1H, d, B of ABq, J=12.13 Hz), 3.56 (1H, d, A ofABq, J=18.09 Hz), 3.22 (1H, d, B of ABq, J=17.74 Hz), 1.99 (3H, s), 1.47(9H, s); high-resolution mass spectrum for [C₁₆H₁₈NO₇SNa₂]⁺, i.e.[M+Na]⁺, m/z calcd 414.0600, found 414.0604.

Example 31 Sodium Salt of 7-[(Z)-acetylmethylene]cephlosporanic Acid(6h)

This compound was prepared from the corresponding ester 4h (0.4 g, 0.84mmol) as described for example 29 to give a yellow fluffy solid (217 mg,78%). R_(f)=0.8 in 5% EtOH in water; UV:λ_(max)=235 nm (50 mM phosphatebuffer, pH=7.2), ε=9,031 cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1750, 1600, 1390cm⁻¹; ¹H NMR (d⁶-DMSO) δ 6.68 (1H, s), 5.56 (1H, s), 4.93 (1H, d, A ofABq, J=12.02 Hz), 4.75 (1H, d, B of ABq, J=11.20 Hz), 3.56 (1H, d, A ofABq, J=17.00 Hz), 3.25 (1H, d, B of ABq, J=17.80 Hz), 2.34 (3H, s), 2.00(3H, s); high-resolution mass spectrum for [C₁₃H₁₃NO₆SNa]⁺, i.e. [M+H]⁺,m/z calcd 334.0361, found 334.0360.

Example 32 Sodium Salt of 7-[(Z)-hydroxymethylmethylene]cephlosporanicAcid (6j)

This compound was prepared from the corresponding ester 4j (0.15 g, 0.32mmol) as described for example 29 to give a white fluffy solid (110 mg,81%). R_(f)=0.86 in water; UV:λ_(max)=222 nm (50 mM phosphate buffer,pH=7.2), ε=8,768 cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1750, 1600, 1390 cm⁻¹; ¹HNMR (d⁶-DMSO) δ 6.32 (1H, s), 5.27 (1H, s), 4.90 (1H, d, A of ABq,J=12.29 Hz), 4.68 (1H, d, B of ABq, J=12.61 Hz), 4.15 (2H, s), 3.45 (1H,d, A of ABq, J=17.46 Hz), 3.22 (1H, d, B of ABq, J=16.80 Hz), 1.97 (3H,s); high-resolution mass spectrum for [C₁₂H₁₃NO₆SNa]⁺, i.e. [M+H]⁺, m/zcalcd 322.0361, found 322.0348.

Example 33 Sodium of Salt of7-[(Z)-N-methoxy-N-methylaminocarbonylmethylene]cephlosporanic Acid (6k)

This compound was prepared from the corresponding ester 4k as describedfor example 29 to give yellow a fluffy solid (yield=55%). R_(f)=0.81 in5% EtOH in water; UV:λ_(max)=231 nm (50 mM phosphate buffer, pH=7.2),ε=11,300 cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1720, 1600, 1390 cm⁻¹; ¹H NMR(d⁶-DMSO) δ 6.84 (1H, s), 5.44 (1H, s), 4,89 (1H, d, A of ABq, J=11.88Hz), 4.72 (1H, d, B of ABq, J=11.90 Hz), 3.72 (3H, s), 3.51 (1H, d, A ofABq, J=17,96 Hz), 3.17 (3H, s), 3.15 (1H, d, B of ABq, J=17.54 Hz), 1.98(3H, s)

Example 34 Sodium Salt of 7-[(E)-bromomethylene]cephalosporanic Acid(6l)

This compound was prepared from the corresponding ester 4l (0.4 g, 0.78mmol) as described for the compound in example 26 to yield a whitefluffy solid (192 mg, 67%). R_(f)=0.77 in 10% EtOH in water;UV:λ_(max)=243 nm (50 mM phosphate buffer, pH=7.2), ε=10,478cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1730, 1600, 1390 cm⁻¹; ¹H NMR (d⁶-DMSO) δ7.21 (1H, s), 5.28 (1H, s), 4.93 (1H, d, A of ABq, J=11.83 Hz), 4.70(1H, d, B of ABq, 11.76 Hz), 3.48 (1H, d, A of ABq, J=17.60 Hz), 3.21(1H, d, B of ABq, J=17.31 Hz), 1.98 (3H, s); high-resolution massspectrum for [C₁₁H₉NO₅SBrNa₂]⁺, i.e. [M+Na]⁺, m/z calcd 391.9178, found391.9180.

Example 35 Sodium Salt of 7-[(Z)-t-butylmethylene]cephalosporanic Acid(6m)

This compound was prepared from the corresponding ester 4m (0.4 g, 0.81mmol) as described for the compound in example 26 to obtain a whitefluffy solid (105 mg, 37%). R_(f)=0.55 in 10% EtOH in water;UV:λ_(max)=228 nm (50 mM phosphate buffer, pH=7.2), ε=12,760cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1730, 1600, 1390 cm⁻¹; ¹H NMR (d⁶-DMSO) δ5.98 (1H, s), 5.00 (1H, s), 4.90 (1H, d, A of ABq, J=11.56 Hz), 4.68(1H, d, B of ABq, J=11.78 Hz), 3.43 (1H, d, A of ABq, J=17.78 Hz), 3.17(1H, d, B of ABq, J=17.45 Hz), 1.98 (3H, s), 1.17 (9H, s);high-resolution mass spectrum for [C₁₅H₁₉NO₅SNa]⁺. i.e. [M+H]⁺, m/zcalcd 348.0877, found 348.0870.

Example 36 Sodium Salt of 7-[dibromomethylene]cephalosporanic AcidSulfone (7a)

This compound was prepared from the corresponding ester 5a (0.3 g, 0.5mmol) as described in the compound in example 26 to give a white fluffysolid (110 mg, 48%). R_(f)=0.83 in 10% EtOH in water; UV: λ_(max)=260 nm(50 mM phosphate buffer, pH=7.2), ε=12,535 cm⁻¹·mol⁻¹·l; IR (KBr) 2950,1740, 1600, 1390, 1330, 1130 cm⁻¹; ¹H NMR (d⁶-DMSO) δ 5.89 (1H, s), 4.94(1H, d, A of ABq, J=12.27 Hz), 4.66 (1H, d, B of ABq, J=12.43 Hz), 4.12(1H, d, A of ABq, J=18.29 Hz), 3.84 (1H, d, B of ABq, J=17.28 Hz), 1.99(3H, s); high-resolution mass spectrum for [C₁₁H₉NO₇SBr₂Na]⁺, i.e.[M+H]⁺, m/z calcd 479.8361, found 479.8349.

Example 37 Sodium Salt of 7-[dichloromethylene]cephalosporanic AcidSulfone (7b)

This compound was prepared from the corresponding ester 5b (0.4 g, 0.76mmol) as described in the compound in example 15 to give a white fluffysolid (yield=50%). R_(f)=0.84 in 10% EtOH in water; UV: λ_(max)=245 nm(50 mM phosphate buffer, pH=7.2), ε=16,679 cm⁻¹·mol⁻¹·l; IR (KBr) 2950,1730, 1600, 1390, 1330, 1130 cm⁻¹; ¹H NMR (d⁶-DMSO) δ 5.99 (1H, s), 4.93(1H, d, A of ABq, J=12.60 Hz), 4.64 (1H, d, B of ABq, J=12.30 Hz), 4.15(1H, d, A of ABq, J=17.57 Hz), 3.90 (1H, d, B of ABq, J=18.16 Hz), 1.97(3H, s); high-resolution mass spectrum for [C₁₁H₈NO₇SCl₂Na₂]⁺, i.e.[M+Na]⁺, m/z calcd 413.9191, found 413.9197.

Example 38 Sodium Salt of 7-[(E)-benzylidene]cephalosporanic AcidSulfone (7c)

This compound was prepared from the corresponding ester 5c (300 mg, 0.55mmol) as described for the compound in example 26 to give title compoundas a white fluffy solid (30 mg, 13% yield). R_(f)=0.70 in 5% EtOH inwater; UV: λ_(max)=308 (50 mM phosphate buffer, _(p)H=7.2), ε=15515cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1710, 1600, 1390, 1330, 1130 cm⁻¹; ¹H NMR(d⁶-DMSO) δ 8.05(2H, m), 7.47 (3H, m), 6.93 (1H, s), 5.72 (1H, s), 4.95(1H, d, A of ABq, J=12.30 Hz), 4.67 (1H, d, B of ABq, J=12.19 Hz), 4.12(1H, d, A of ABq, J=17.60 Hz), 3.79 (1H, d, B of ABq, J=17.63 Hz), 2.00(3H, s); high-resolution mass spectrum for [C₁₇H₁₅NO₇SNa]⁺, i.e. [M+H]⁺,m/z calcd 400.0463, found 400.0451.

Example 39 Sodium Salt of 7-[(Z)-benzylidene]cephalosporanic AcidSulfone (7d)

This compound was prepared from the corresponding ester 5d (250 mg, 0.46mmol) as described in the compound in example 26 to give title compoundas a white fluffy solid (77 mg, 42% yield). R_(f)=0.80 in 5% EtOH inwater; UV: λ_(max)=302 (50 mM phosphate buffer, _(p)H=7.2), ε=20543cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1740, 1600, 1390, 1330, 1130 cm⁻¹; ¹H NMR(d⁶-DMSO) δ 7.79 (2H, m), 7.44 (4H, m), 6.34 (1H, s), 4.95 (1H, d, A ofABq, J=12.05 Hz), 4.70 (1H, d, B of ABq, J=11.75 Hz), 4.12 (1H, d, A ofABq, J=17.73 Hz), 3.88 (1H, d, B of ABq, J=17.54 Hz), 2.02 (3H, s);high-resolution mass spectrum for [C₁₇H₁₅NO₇SNa]⁺, i.e. [M+H]⁺, m/zcalcd 400.0463, found 400.0464.

Example 40 Sodium Salt of 7-[(Z)-(2′-pyridyl)methylene]cephalosporanicAcid Sulfone (7e)

This compound was prepared from the corresponding ester 5e as describedin as described for example 29 as a pale yellow fluffy solid(yield=67%). R_(f)=0.78 in 10% EtOH in water; UV: λ_(max)=301 nm (50 mMphosphate buffer, pH=7.2), ε=8,624 cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1720,1600, 1390, 1330, 1130 cm⁻¹; ¹H NMR (d⁶-DMSO) δ 8.59 (1H, d), 7.88 (1H,t), 7.72 (1H, d), 7.42 (2H, m), 6.22 (1H, s), 4.92 (1H, d, A of ABq,J=11.43 Hz), 4.72 (1H, B of ABq, J=10.87 Hz), 4.19 (1H, D, A of ABq,J=17.58 Hz), 3.77 (1H, d, B of ABq, J=18.45 Hz), 2.00 (3H, s);high-resolution mass spectrum for [C₁₆H₁₃N₂O₇SNa₂]⁺, i.e. [M+Na]⁺, m/zcalcd 423.0239, found 423.0227.

Example 41 Sodium Salt of 7-[(Z)-t-butoxymethylene]cephalosporanic AcidSulfone (7g)

This compound was prepared from the corresponding ester 5g (0.3 g, 0.53mmol) as described in as described for example 29 to give a white fluffysolid (163 mg, 73%). R_(f)=0.74 in 5% EtOH in water; UV:λ_(max)=226 nm(50 mM phosphate buffer, pH=7.2), ε=18,171 cm⁻¹·mol⁻¹·l; IR (KBr) 2950,1720, 1600, 1390, 1330, 1130 cm⁻¹; ¹H NMR (d⁶-DMSO) δ 6.52 (1H, s), 5.97(1H, s), 4.97 (1H, d, A of ABq, J=12.36 Hz), 4.72 (1H, d, B of ABq,J=12.63 Hz), 4.16 (1H, d, A of ABq, J=12.63 Hz), 3.79 (1H, d, B of ABq,J=18.21 Hz), 1.99 (3H, s), 1.45 (9H, s). high-resolution mass spectrumfor [C₁₆H₁₉NO₉SNa]⁺, i.e. [M+H]⁺, m/z calcd 424.0678, found 424.0684.

Example 42 Sodium Salt of 7-[(Z)-hydroxymethylmethylene]cephalosporanicAcid Sulfone (7j)

This compound was prepared from the corresponding ester 5j (0.2 g, 0.4mmol) as described in as described for example 29 to give a white fluffysolid (130 mg, 91.5%). R_(f)=0.90 in water; λ_(max)=223 nm (50 mMphosphate buffer, pH=7.2), ε=9,428 cm⁻¹·mol⁻¹·l; IR (KBr) 2950, 1750,1600, 1390, 1330, 1130 cm⁻¹; ¹H NMR (d⁶-DMSO) δ 6.54 (1H, s), 5.74 (1H,s), 4.89 (1H, d, A of ABq, J=12.05 Hz), 4.66 (1H, d, B of ABq, J=11.93Hz), 4.14 (2H, s), 4.08 (1H, d, A of ABq, J=19.29 Hz), 3.73 (1H, d, B ofABq, J=18.25 Hz), 1.99 (3H, s); high-resolution mass spectrum for[C₁₂H₁₃NO₈SNa]⁺, i.e. m/z calcd 354.0260, found 354.0274.

Example 43 Sodium Salt of 7-(E)-[bromomethylene]cephalosporanic AcidSulfone (7l)

This compound was prepared from the corresponding ester 5l (0.3 g, 0.55mmol) as described in the compound in example 26 to yield a white fluffysolid (128 mg, 58%). R_(f)=0.88 in 10% EtOH in water; UV: λ_(max)=246 nm(50 mM phosphate buffer, pH=7.2), ε=10,856 cm⁻¹·mol⁻¹·l; IR (KBr) 2950,1730, 1600, 1390, 1330, 1130 cm⁻¹; ¹H NMR (d⁶-DMSO) δ 7.44 (1H, s), 5.79(1H, s), 4.96 (1H, d, A of ABq, J=12.30 Hz), 4.68 (1H, d, A of ABq,J=12.28 Hz), 4.03 (1H, d, A of ABq, J=18.53 Hz), 3.82 (1H, d, B of ABq,J=17.69 Hz), 2.00 (3H, s); high-resolution mass spectrum for[C₁₁H₁₀NO₇SBrNa]⁺, i.e. [M+H]⁺, m/z calcd 401.9256, found 401.9245.

Example 44 Sodium Salt of 7-[(Z)-t-butylmethylene]cephalosporanic AcidSulfone (7m)

This compound was prepared from the corresponding ester 5m (0.34 g, 0.65mmol) as described in the compound in example 26 to yield a white fluffysolid (2.0 g, 82%). R_(f)=0.79 in 10% EtOH in water; UV: λ_(max)=228 nm(50 mM phosphate buffer, pH=7.2), ε=14,215 cm⁻¹·mol⁻¹·l; IR (KBr) 2950,1730, 1600, 1390, 1330, 1130 cm⁻¹; ¹H NMR (d⁶-DMSO) δ 6.08 (1H, s), 5.50(1H, s), 4.91 (1H, d, A of ABq, J=12.24 Hz), 4.65 (1H, d, B of ABq,J=12.26 Hz), 4.06 (1H, d, A of ABq, J=17.41 Hz), 3.72 (1H, d, B of ABq,J=17.76 Hz), 1.99 (3H, s); high-resolution mass spectrum for[C₁₅H₁₈NO₇SNa]⁺, i.e. [M+H]⁺, m/z calcd 380.0775, found 380.0770.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A compound of formula (1)

wherein n is 0; R₁ and R₂ are the same or different and are selectedfrom the group consisting of a) hydrogen; b) linear or branchedC₁-C₁₀-alkyl; c) halogen; d) C₁₋₁₀-alkoxy; e) C₂₋₁₀-alkanoyloxy; f)C₂₋₁₀-alkene; g) C₂₋₁₀-alkene substituted with one or more groupsselected from the group consisting of chlorine, fluorine, bromine orphenyl; h) C₁₋₁₀-alkoxycarbonyl; I) C₁₋₁₀-alkoxycarbamido; j)N—C₁₋₁₀-alkoxy-N—C₁₋₁₀-alkylaminocarbonyl; k) halo-C₁₋₁₀-alkyl; l)C₆₋₁₀-aryl; m) C₆₋₁₀-aryl; substituted with one or more groups selectedfrom the group consisting of ethyl, n-propyl, isopropyl, amino,methylamino and dimethylamino; n) a C₂-C₁₀ heterocycle having from 1-3heteroatoms selected from the group consisting of O, N, and S; o) —COOHor —COOY, wherein Y is pharmaceutically acceptable cation; and, p) COMe;R₃ is —CH₂M where M is selected from the group consisting of a)C₂₋₁₀-acyloxy; and b) C₂₋₁₀-acyloxy substituted with one or more groupsselected from the group consisting of —COOH, aminophenyl, phenyl,C₁₋₆-alkyl, chlorine, bromine or fluorine; R₄ is selected from the groupconsisting of a) hydrogen; and, b) pharmaceutically acceptable cations.2. A compound of claim 1 which is 7-[dibromomethylene]cephalosporanicacid; or a pharmaceutically acceptable salt thereof.
 3. A compound ofclaim 1 which is 7-[dichloromethylene] cephalosporanic acid; or apharmaceutically acceptable salt thereof.
 4. The compound of claim 1,wherein R₂ is hydrogen and R₁ is selected from the group consisting oft-butyl, phenyl, pyridyl, COMe and N-methyl-N-methoxyaminocarbonyl. 5.The compound of claim 1, wherein R₂ is hydrogen, and R₁ is CO₂-t-Bu. 6.The compound of claim 1, wherein R₁ and R₂ are the same and are selectedfrom the group consisting of bromine and chlorine.
 7. The compound ofclaim 1, wherein R₁ is hydrogen, and R₂ is bromine.
 8. A pharmaceuticalcomposition, comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 9. A compound of claim 1 which is:7-[dibromomethylene] cephalosporanic acid;7-[dichloromethylenie]cephalosporanic acid;7-[(Z)-(2′-pyridyl)methylene] cephalosporanic acid;7-[(Z)-methoxycarbonylmethylene]cephalosporanic acid;7-[(Z)-t-butoxycarbonylmethylene]cephalosporanic acid;7-[(Z)-acetylmethylene] cephalosporanic acid;7-[(Z)-N-methoxy-N-methylaminocarbonylmethylene]cephalosporanic acid;7-[(E)-bromomethylene]cephalosporanic acid; or 7-[(Z)-t-butylmethylene]cephalosporanic acid.
 10. A compound of formula (1)

wherein n is 1; R₁ and R₂ are the same or different and are selectedfrom the group consisting of a) hydrogen; b) linear or branchedC₁-C₁₀-alkyl; c) halogen; d) hydroxy-C₁₋₁₀-alkyl; e) C₁₋₁₀-alkoxy; f)C₂₋₁₀-alkanoyloxy; g) C₂₋₁₀-alkene; h) C₂₋₁₀-alkene substituted with oneor more groups selected from the group consisting of chlorine, fluorine,bromine or phenyl; i) C₁₋₁₀-alkoxycarbonyl; j) C₁₋₁₀-alkoxycarbamido; k)N—C₁₋₁₀-alkoxy-N—C₁₋₁₀-alkylaminocarbonyl; l) halo-C₁₋₁₀-alkyl; m)C₆₋₁₀-aryl; n) C₆₋₁₀-aryl substituted with one or more groups selectedfrom the group consisting of ethyl, n-propyl, isopropyl, amino,methylamino and dimethylamino; o) a C₂-C₁₀ heterocycle having from 1-3heteroatoms selected from the group consisting of O, N, and S; and, p)—COOH or —COOY, wherein Y is pharmaceutically acceptable cation; R₃ is—CH₂M where M is a) C₂₋₁₀-acyloxy; or b) C₂₋₁₀-acyloxy substituted withone or more groups selected from the group consisting of —COOH,aminophenyl, phenyl, C₁₋₆-alkyl, chlorine, bromine or fluorine; and R₄is selected from the group consisting of a) hydrogen; and, b)pharmaceutically acceptable cations.
 11. The compound of claim 10wherein R₁ is phenyl, pyridyl or CO₂-t-butyl; and R₂ is hydrogen. 12.The compound of claim 10 wherein R₁ is 2-pyridyl; R₂ is hydrogen; M isacetoxy; and R₄ is sodium.
 13. A pharmaceutical composition, comprisinga compound of claim 10 and a pharmaceutically acceptable carrier.